It has been established that many human tumors contain a significant hypoxic fraction of cells [Kennedy et al., Int. J. Radiat. Oncol. Biol. Phys., 1997, 37, 897; Movsas et al., Urology, 1999, 53, 11]. The presence of hypoxic cells arises because the extravascular transport (EVT) of oxygen is compromised due to an inefficient microvascular system within the tumor, which leads to large intercapillary distances and variable blood flow. Reduction of oxygen tension in tumors leads to radio-resistance. This reduction of oxygen tension causes up to a three-fold increase in radiation dose being required to kill anoxic tumour cells. A link has been identified between the presence of tumour hypoxia and failure of local control by radiation therapy [Brizel et al., Radiother. & Oncol., 1999, 53, 113].
This phenomenon of tumour hypoxia has been exploited in the development of ‘bioreductive drugs’ [Brown et al., Semin. Radiat. Oncol. 1966, 6, 22; Denny et al., Br. J. Cancer, 1996, 74 (Suppl. XXVII) 32; Stratford & Workman, Anti-Cancer Drug Des., 1998, 13, 519]. These agents are prodrugs that are selectively activated by enzymatic reduction in hypoxic cells, resulting in formation of a cytotoxin.
The 3-amino-1,2,4-benzotriazine 1,4-dioxides have been developed as bioreductive drugs for cancer therapy [Brown, Br. J. Cancer, 1993, 67, 1163-1170; Minchinton et al., Int. J. Radiat. Oncol. Biol. Phys. 1992, 22, 701-705 Kelson et al., Anti-Cancer Drug Des., 1998, 13, 575; Lee et al., WO 91/04028, April 1991]. The lead compound of this class, tirapazamine (TPZ; SR 4233), is undergoing clinical trials in combination with radiotherapy and various chemotherapeutics, notably cisplatin [Denny & Wilson, Exp. Opin. Invest. Drugs, 2000, 9, 2889]. TPZ is activated by one electron reductases [Patterson et al., Anti-Cancer Drug Des. 1998 13, 541; Denny & Wilson, Exp. Opin. Invest. Drugs, 2000, 9, 2889] to form a radical that may be oxidized back to TPZ by molecular oxygen under aerobic conditions. Under hypoxic conditions the radical spontaneously generates an oxidizing radical(s) R• (considered to be the hydroxyl radical [Daniels and Gates, J. Am. Chem. Soc., 1996, 118, 3380-3385], and/or a benzotriazinyl radical [Anderson et al., J. Am. Chem. Chem. 2003, 125, 748-756]) which interact with DNA (and/or topoisomerase II)[Peters and Brown, Cancer Res., 2002, 62, 5248-5253] to cause double-strand breaks and these correlate with cytotoxicity [Dorie et al., Neoplasia, 1999, 1, 461]. These features are illustrated in Scheme 1.

There have been only limited structure-activity studies on analogues of TPZ. Kelson et al. [Anti-Cancer Drug Design, 1998, 13, 575], Zeman et al. [Int. J. Radiat. Oncol. Biol. Phys., 1989, 16, 977-981] and Minchinton et al. [Int. J. Radiat Oncol. Biol. Phys., 1992, 22, 701-705 ] disclosed compounds of type I,
where X was H or an electron-withdrawing group, n was 2 or 3, and R was Me or Et. This paper showed that compounds with dialkylaminoalkyl side chains showed variable hypoxic selectivity in vitro. Compounds where X═H and having dialkylamino side chains had a similar hypoxic cytotoxicty ratio to TPZ and comparable or inferior activity to TPZ in vivo.
Hay and Denny [Tet. Lett., 2002, 43, 9569], Minchinton et al. [Int. J. Radiat. Oncol. Biol. Phys., 1992, 22, 701-705 ] and Kelson et al. [Anti-Cancer Drug Design, 1998, 13, 575] described compounds of type II,
where X is H or hydroxyalkyl and R is OH or OMe. Kelson et al. [Anti-Cancer Drug Design, 1998, 13, 575] and Minchinton et al., [Int. J. Radiat. Oncol. Biol Phys., 1992, 22, 701-705 ] suggested that 3-alkyl compounds (X═H, n=1,2 or 3, R═H and X═H, n=2, R═OMe) were comparable to TPZ in vivo.
Finally, Hay et al. [Hay et al., J. Med. Chem. 2003, 46, 169] showed, for compounds of type III,
that there is an optimum range of one-electron reduction potential [E(1)] (between ca. −450 to −510 mV) for in vitro hypoxic selectivity. However, there was no clear relationship between the electron-withdrawing capability of the 7-substituent on the benzo ring and the reported biological activity.
Throughout this specification several abbreviations are used that require explanation and the following glossary is provided.                IC50: The concentration of drug (in micromolar, μM) to reduce cell numbers to 50% of those of control cell cultures grown under the same conditions but not exposed to drug.        HCR: Hypoxic cytotoxicity ratio (the ratio of drug concentrations under aerobic and hypoxic conditions to produce equal cell survival (50%) determined by proliferation assay)        Kmet: First order rate constant for metabolism of a drug estimated at the C10 (see below)        C10: the concentration required to produce one log of cell kill after exposure of cells to drug for one hour in clonogenic assays described in the methods (below).        PK: Pharmacokinetics. (Description of the variation in drug concentration with time (i.e. exposure) in a specified compartment or position within a tissue)        PD: Pharmacodynamics. (Description of the biological response to a drug)        PK/PD Model: Mathematical description of the relationship between drug exposure (PK) and biological response (PD).Drawbacks of TPZ        
Despite its advancement in clinical trials, several limitations of TPZ have been identified, including its relatively low solubility and poor therapeutic ratio. It is clear that the therapeutic ratio of TPZ in both preclinical (murine and human tumours) and clinical studies is low, with substantial toxicity at efficacious doses [Rischin et al., Proc. Am. Soc. Clin. Oncol. 2003, 22, 495-496] and that there is a need for more tumour selective analogues. Preclinical studies have identified extravascular transport (EVT) as a factor that limits activity of TPZ against hypoxic cells in tumours [Durand & Olive Radiat. Oncol. Investig. 1997, 5, 213; Durand & Olive, Int. J. Radiat. Oncol. Biol. Phys. 1992, 22, 689; Hicks et al, Int. J. Radiat. Oncol. Biol. Phys. 1998, 42, 641; Hicks et al, Cancer Res. 2003, 63, 5970; Kyle & Minchinton, Cancer Chemother. Pharmacol. 1999, 43, 213].
The EVT problem is thought to be particularly severe for bioreductive drugs, such as TPZ, for two reasons:                1. The target hypoxic cells are generally those most distant from the blood vessels        2. The metabolism of the bioreductive drug in the hypoxic tumour tissue will cause a continuously falling gradient of drug concentration through both the oxic and hypoxic tumour tissue which may not be overcome even with long infusion times.        
However the same bioreductive metabolism which limits drug transport is also responsible for the cytotoxic effect of the drug [Baker et al. Cancer Res., 1988, 48, 5947-5952; Siim et al, Br. J. Cancer 1996, 73, 952]. These competing effects of drug metabolism on EVT and cytotoxicity have been investigated using the multicellular layer model [Hicks et al, Int. J. Radiat. Oncol. Biol. Phys. 1998, 42, 641], as illustrated in FIG. 1. Parameters determined by this model, together with single cell experiments to determine cytotoxicity and rates of metabolism [Hicks et al, Cancer Res. 2003, 63, 5970] and the oxygen dependence of cytotoxicity [Hicks et al., Radiat. Res. 2004, 161, 656 ] are used in a pharmacokinetic/pharmacodynamic (PK/PD) model of cell killing in tumour tissue (as illustrated in FIG. 2). The model and results obtained have demonstrated the need to optimise (rather than maximise) the rate of bioreductive metabolism. FIG. 2 illustrates that high rates of metabolism will limit drug penetration and thus reduce cell kill in the hypoxic region, as well as decrease the differential in killing of hypoxic cells compared to well oxygenated cells. This is consistent with experimental results where high rates of metabolism limited activity in anoxic V79 multicellular spheroids [Durand & Olive Int. J. Radiat. Oncol. Biol. Phys. 1992, 22, 689] and anoxic HT29 MCL [Hicks et al., Cancer Res. 2003, 63, 5970], and resulted in a reduced hypoxic cytotoxic differential in SiHa human cervical tumours grown in SCID mice [Durand & Olive, Radiat. Oncol. Investig. 1997, 5, 213].
The above PK/PD model for TPZ, developed by Hicks et al., can be described as a distributed parameter model because it considers explicitly the spatial variation in parameter values (in other words it describes PK, and PD, as a function of position in tumour tissue). The main aspects of this distributed parameter PK/PD model have been disclosed in several publications (Hicks et al., Int. J. Radiat. Oncol. Biol. Phys. 1998, 42, 641; Hicks et al., Cancer Res. 2003, 63, 5970; Hicks et al., Proc Am. Assoc. Cancer Res, 2003, Abstract #4561; Wilson et al., Proc Am. Assoc. Cancer Res, 2003, Abstract #4570).
The key PK/PD relationship, as determined by investigating TPZ metabolism to its reduction product SR 4317 and cell killing as a function of TPZ concentration and time in anoxic stirred suspensions of HT29 colon carcinoma cells (Hicks et al., Cancer Res., 2003), is described by:
            Eqn      ⁢                          ⁢      1      ⁢              :              ⁢                  ⁢                  -                            ⅆ          log                ⁢                                  ⁢        SF                    ⅆ        t              =      γ    ⁢                  ⁢    C    ⁢                  ⅆ        M                    ⅆ        t            
This relationship shows that the rate at which cells are killed (on a log scale; SF=surviving fraction) is proportional to the rate of bioreductive drug metabolism (M, the amount of drug metabolised per unit intracellular volume) and to the drug concentration, C. The constant of proportionality, γ, is a cell-line dependent parameter determined by fitting the model to clonogenic survival curves where drug concentrations are measured simultaneously. Under conditions of constant TPZ concentration, this approximates a concentration2×time dependence of log cell kill on TPZ exposure.
In order to describe PK/PD as a function of position in tumours, the above PK/PD model is extended to a spatially resolved (distributed parameter) model by incorporating the EVT properties (diffusion coefficient and rate of metabolism) of TPZ. In addition, because oxygen concentration in tumours varies as a function of distance from blood vessels, it is necessary to describe the relationship between O2 concentration and rate of TPZ metabolism.
Simulation of TPZ diffusion into a tumour—in one dimension is illustrated in (FIG. 3A. An O2 concentration gradient in the one dimension planar tissue can be calculated numerically by solving the reaction-diffusion equation:
      Eqn    ⁢                  ⁢    2    ⁢          :        ⁢                                      ∂                  C                      O            2                                      ∂        t              =                            D          O2                ⁢                                            ∂              2                        ⁢                          C                              O                2                                                          ∂                          x              2                                          -                          ⁢                        ∂                      C                          O              2                                                ∂          t                    where CO2 is the oxygen concentration in μM at position x and time t, using the diffusion coefficient and rate of metabolism in R3230Ac tumors (Dewhirst et al., Cancer Res., 1994, 54, 3333; Secomb et al., Adv. Exp. Med. Biol. 1998, 454, 629) assuming an arteriolar input oxygen concentration of [O2]=50 μM (38 mm Hg).
TPZ concentrations in the one dimensional tissue are calculated numerically from the reaction diffusion equation:
      Eqn    ⁢                  ⁢    3    ⁢          :                            ∂        C                    ∂        t              =                            D          MCL                ⁢                                            ∂              2                        ⁢            C                                ∂                          x              2                                          -                          ⁢              ϕ        ⁢                                  ⁢                  f          ⁡                      (                          [                              O                2                            ]                        )                          ⁢                              ∂            M                                ∂            t                              where C is the concentration of drug at position x and time t, DMCL is the diffusion coefficient of drug in the multicellular layers, and φ is the cell volume fraction of the multicellular layer. Oxygen inhibits TPZ metabolism and this effect can be calculated according to the equation:
      Eqn    ⁢                  ⁢    4    ⁢          :                    f      ⁡              (                  [                      O            2                    ]                )              =          (                        K                      O            2                                                K                          O              2                                +                      [                          O              2                        ]                              )      where KO2 is the O2 concentration required for half maximal inhibition of TPZ metabolism.
The above relationships (Eqn 1-4) define a PK/PD model for TPZ. The output of the model depends on the plasma PK of free drug (drug not bound to plasma proteins), which provides the input to the extravascular compartment, and on the geometry of the transport problem. Using this model the surviving fraction at each point in the tissue is calculated and the average log cell kill in the target hypoxic region evaluated. This is illustrated in FIG. 2 for TPZ at its maximum tolerated in mice, together with simulations for a faster diffusing drug and an analogue with higher rates of metabolic activation.
This PK/PD model can be further extended to take into account the effects of diffusion in a three dimensional-3D microvascular environment, such as irregular vascular geometry, blood flow heterogeneity, and loss of oxygen and drug during transit through a capillary network. Such a 3D network is illustrated in FIG. 3B and further described in (Hicks et al., Proc Am. Assoc. Cancer Res, 2003, Abstract #4561; Wilson et al., Proc Am. Assoc. Cancer Res, 2003, Abstract #4570). In this model similar equations as Eqns 1-4 are solved numerically using a Green's function method similar to that used for oxygen alone (Secomb et al., Adv. Exp. Med. Biol. 1998, 454, 629).
In order to test the validity of this PK/PD model as a tool for predicting the antitumour activity of TPZ analogues, the inventors determined the key PK/PD parameters for 13 TPZ analogues, and TPZ itself, for the HT29 human colon carcinoma cell line. These parameters were then used to calculate the expected killing in hypoxic regions of HT29 tumours following a single intraperitoneal dose of the compounds at their maximum tolerated dose. The results of this calculation were then compared with measured killing of hypoxic cells in HT29 tumours, determined by administering the compounds immediately (within 5 min) after gamma irradiation (cobalt 60, 20 Gy) to sterilise oxygenated cells in the tumours. Tumours were removed 18 hours later and the number of surviving cells determined by clonogenic assay. The results for one compound 3 are shown in FIG. 5. The measured PK/PD parameters, model prediction (using the above 3D microvascular network), and experimentally determined hypoxic cell kill is shown in Table 1, and the relationship between predicted and measured cell kill in FIG. 4. The prediction is statistically highly significant (p=0.001, Fisher exact test). Comparing the magnitude of predicted and observed response (FIG. 4) also shows a highly significant linear correlation (R2=0.94, p<0.001 for a non-zero slope). If the extravascular transport component was excluded from the model, the R2 for this regression was only 0.28 and the relationship was not statistically significant.
TABLE 1Parameters of the PK/PD model for 14 benzotriazine di-N-oxides, modelprediction of hypoxic cell killing in tumours, and measured hypoxic cell killing.DMCLkmetγAUCflog killlog killStat.Cmpdcm2s−1min−1μM2μM.hr(Pred)(Meas)SESignif.A3.97 × 10−70.602.21 × 10−5148.11.2481.1700.149<0.01B5.43 × 10−70.201.64 × 10−5188.71.9232.1650.149<0.01C8.70 × 10−73.923.71 × 10−51.90.005—0.0070.184nsD6.69 × 10−710.032.25 × 10−60.80.000—0.1540.064nsE2.13 × 10−71.001.40 × 10−615.60.000—0.0280.083nsF5.09 × 10−72.546.84 × 10−53.50.0110.1960.062nsG1.71 × 10−64.893.90 × 10−57.20.0480.2480.072nsH3.99 × 10−71.521.10 × 10−469.00.9851.0010.142<0.01I2.61 × 10−61.916.52 × 10−6140.61.1160.8860.132<0.01J2.09 × 10−62.012.90 × 10−479.00.8900.8000.149<0.05K6.17 × 10−718.339.86 × 10−30.0420.0000.0090.102nsL3.82 × 10−79.181.30 × 10−33.60.078−0.0510.039nsM9.80 × 10−89.133.04 × 10−30.90.0560.1380.100nsN1.05 × 10−74.742.39 × 10−21.30.0920.0270.121nsAUCf = AUC of free drugLog kill (Pred) = -log10 (hypoxic cell surviving fraction) predicted by the modelLog kill (Meas) = -log10 (hypoxic cell surviving fraction) measured in tumoursSE = standard error of log kill (Meas).Stat. Signif. = statistical significance of log kill (Meas).ABCDEFGHIJKLMN
It is established by these studies that extravascular transport is a determinant of in vivo cytotoxicity and selectivity of benzotriazine di-N-oxides. These results confirm that the measurement of parameters such as IC50 and HCR in cell culture, alone, do not provide a reliable prediction for activity against hypoxic cells in tumours.
However, despite the elegance of these models and the highly statistically significant results achieved, one of the inherent difficulties with this approach is that it requires complex computational methods, and a detailed knowledge of a microvascular network geometry and blood flow. This information is not generally available.
It is therefore an object of the present invention to overcome some of these complexities by providing a simplified set of characteristics that can be used to select 1,2,4 benzotriazine 1,4 dioxide compounds (TPZ analogues) with therapeutic activity against hypoxic cells in human tumour xenografts, and to provide a method by which these characteristics can be assessed with minimal or no testing of the compounds in animals and to further provide a novel class of TPZ analogues with predicted improved in vivo activity against tumours, relative to TPZ, or to at least provide the public with a useful choice.